The development of methodologies for the early detection of genetic mutations is an important issue in the prevention and treatment of malignancy. Current knowledge of the association of specific genetic alterations with the development of certain types of tumors enables an approach to the early detection of cancer long before histologic or pathologic evidence indicates the development of neoplastic tumor growth. The identification of genetic alterations as biological markers allows for early diagnosis, which in turn may dictate a particular regimen of treatment to prevent subsequent tumor development.
The multistep process of transformation is thought to be directed by an accumulation of specific dominant and recessive genetic lesions. For example, one frequent dominant event in somatic cell cancers has been found to be an activating mutation in a member of the ras gene family. Strong support exists for the concept that ras oncogenes are causative players in the multistep process of tumorigenesis. Ras gene mutations occur in approximately 15% of human tumors, however their incidence appears to vary according to tumor type. Specifically, mutations in the human K-ras gene have been reported to be as high as 90% in carcinomas of the pancreas, 60% in adenocarcinomas of the lung, and 50% in adenocarcinomas of the colon. In certain tumor types, K-ras gene mutations present themselves as early events in the tumorigenic pathway, and evidence suggests that they are more prevalent during the later stages of tumor development.
The most frequently activated position of the K-ras gene in human tumors has been found to be codon 12. Therefore, by way of example, the detection of mutations at this position could be of critical importance in many aspects of research and diagnostic application. Furthermore, the clinical application of the assay for detection of mutations in biological samples could be of great prognostic value, and could assist in evaluating early courses of patient intervention.
Presently, the detection of mutant genes has been accomplished through the use of the polymerase chain reaction (PCR), a quick and simple in-vitro reaction through which adequate amounts of a specific gene region can be generated for subsequent analysis. Amplified DNA fragments have been analyzed for the presence of point mutations employing one of several technical approaches briefly outlined below:
1. Single-strand conformation polymorphism analysis is a method for analyzing DNA for nucleotide substitutions. In this method, amplified material is denatured to create single-stranded DNA and separated on a native polyacrylamide gel under conditions that enable distinction between single strands of normal and mutant alleles, each migrating at a different rate. This technique can be used to identify alterations in any given gene without requiring knowledge of the specific site where a mutation has occurred. PA1 2. Sequencing an amplified product of a specific gene is an approach that leads to the direct identification of a mutated site. This approach is the most labor-intensive, yet it provides complete information with respect to the type of mutation and its precise location. PA1 3. Restriction fragment length polymorphism where PCR amplified products are digested with specific restriction enzymes which can selectively digest either a normal or a mutated allele or a particular gene. To obtain higher sensitivity, the RFLP has been modified through the incorporation of a liquid hybridization step in which amplified material is hybridized with a labelled oligonucleotide sequence which is specific for a mutated region prior to separation on PAGE. This approach, also known as high resolution RFLP analysis, eliminates the need for sequencing, but it is limited to the analysis of mutations at a precise location that involves a naturally occurring restriction enzyme site. To overcome this limitation, one can artificially introduce restriction enzyme sites to permit a distinction to be made between normal and mutant alleles where the position of the point mutation does not harbor a naturally occurring site. In this approach, base-pair substitutions are introduced into the primers used for the PCR, yielding a restriction enzyme site only when the primer flanks a specific point mutation. This approach enables the selective identification of a point mutation at a known site of presumably any gene. PA1 4. Enriched PCR is a modification introduced by the inventor (collaborating with others) into the RFLP analysis which permits the detection of a mutant gene even when the mutation is present at very low frequency (i.e. 1 in 10.sup.4 normal alleles). The principle of this approach is to create a restriction enzyme site only with normal sequences, thus enabling selective digestion of normal but not of mutant alleles amplified in a first amplification step. This prevents the non-mutant DNA from further amplification in a second amplification step while, upon subsequent amplification, the mutated alleles are enriched. PA1 5. Mismatched 3' end amplifications is a PCR technique which utilizes 5' primers that have been modified at the 3' end to match only one specific point mutation. This method relies on conditions under which primers with 3' ends complementary to specific mismatches are amplified, whereas wild-type sequences preclude primer elongation. This procedure requires a specific primer for each suspected alteration and must be carried out under rigorous conditions. PA1 (i) a first amplification step comprising amplifying material in first and second genomic duplexes present in the test sample in a first polymerase chain reaction in which upstream and downstream long tail primers, comprising upstream primer and downstream primer nucleotide sequences respectively, DNA polymerase, four different nucleotide triphosphates and a buffer are used in a repetitive series of reaction steps involving template denaturation, primer annealing and extension of annealed primers to form first and second synthesized nucleic acid duplexes. Each of the first synthesized nucleic acid duplexes has an upstream end and a downstream end and consists of a first synthesized strand and a first complementary synthesized strand. Each of the second synthesized nucleic acid duplexes has an upstream end and a downstream end and consists of a second synthesized strand and a second complementary synthesized strand. Each of the first and second synthesized strands has a first end portion comprising the upstream primer nucleotide sequences and a second end portion comprising nucleotide sequences sufficiently complementary to the downstream primer nucleotide sequences to anneal therewith. The first synthesized duplexes have the region with a mutant nucleotide sequence, and the second synthesized duplexes have the region with a wild-type nucleotide sequence. The upstream and downstream long tail primers are selected such that nucleic acid strands formed in the first polymerase chain reaction using the upstream and downstream long tail primers can anneal with short tail primers which do not anneal with any nucleic acid strands in the (first or second) genomic duplexes. The long tail upstream primers are also selected such that the second synthesized duplexes have a restriction site which is not present in the first synthesized duplexes due to the presence in the first synthesized duplexes of the region with the mutant nucleotide sequence. The restriction site is cleavable with a first restriction enzyme. PA1 ii) a digestion step comprising treating at least a portion of the test sample with the first restriction enzyme whereby selectively to cleave the second synthesized duplexes while leaving the first synthesized duplexes uncleaved, PA1 iii) a second amplification step comprising amplifying material that was subjected to restriction enzyme digestion in step (ii) and remained uncleaved. In this amplification step, upstream and downstream short tail primers are used in a second polymerase chain reaction selectively to reamplify material which was synthesized in the first amplification step and was not affected by the restriction enzyme in step (ii) since it harbors a mutation in the specific region of the genome. The upstream and downstream short tail primers are selected such that they anneal with the first synthesized complementary and first synthesized strands respectively but do not anneal with strands of the first or second genomic duplexes whereby the upstream and downstream short tail primers can be used in the second amplification step selectively to amplify material in duplexes formed in the first amplification step but cannot amplify material in the first or second genomic duplexes. Each of the upstream short tail primers are labelled with a first substance that binds tightly with a second substance such that upstream ends of the further synthesized duplexes bind to a supporting surface coated with the second substance. Each of the downstream short tail primers are labelled with a downstream label such that downstream ends of the further synthesized duplexes have the downstream label. The second amplification step is performed in a vessel (e.g. microwell plate; Eppendorf tube) having the supporting surface coated with the second substance such that further synthesized duplexes labelled with the first substance contact and bind to the supporting surface, or the process includes a binding step comprising contacting the test sample with the supporting surface coated with the second substance whereby further synthesized duplexes labelled with the first substance bind thereto. The binding step can be performed, for example, after second stage amplification or after a subsequent digestion with the restriction enzyme. PA1 iv) a second digestion step wherein the test sample is again treated with the first restriction enzyme selectively to cleave synthesized duplexes containing regions having the wild-type sequence; PA1 v) a washing step to remove at least downstream portions of cleaved duplexes from the supporting surface; and PA1 vi) a detection step comprising assaying for the presence of the downstream label on the supporting surface. PA1 a) a first reagent mixture for use in the first amplification step wherein material in the first nucleic acid duplexes is amplified in a polymerase chain reaction with synthesis of a first synthesized duplex having a first synthesized nucleic acid strand and a first complementary synthesized strand. The first reagent mixture comprises upstream and downstream long tail primers. Each of the upstream and downstream long tail primers comprises a complementary primer portion and a non-complementary primer portion. The complementary primer portion of the upstream long tail primers is sufficiently complementary to a first end portion of the first complementary nucleic acid strand to enable the upstream long tail primers to anneal therewith and thereby to initiate synthesis of a nucleic acid extension product using the first complementary nucleic acid strand as a template. The complementary primer portion of the downstream long tail primers is sufficiently complementary to a first end portion of the first genomic strand to enable the downstream primers to anneal therewith and thereby to initiate synthesis of a nucleic acid extension product using the first genomic strand as a template. The non-complementary primer portions of the upstream and downstream long tail primers are not sufficiently complementary to either the first genomic strand or the first complementary nucleic acid strand to anneal with either. The non-complementary primer portions of the respective upstream and downstream long tail primers are positioned on the respective upstream and downstream long tail primers such that a first end portion of the first synthesized strand has nucleotide sequences that are identical to the nucleotide sequences of the non-complementary primer portion of the upstream long tail primers and such that a first end portion of the first complementary synthesized strand has nucleotide sequences that are identical to the nucleotide sequences of the non-complementary primer portion of the downstream primers; and PA1 b) a second reagent mixture for use in the second amplification step comprising upstream and downstream short tail primers. Each of the upstream short tail primers has nucleotide sequences which are sufficiently complementary to the nucleotide sequences in the non-complementary primer portion of the upstream long tail primers to anneal therewith but which are not sufficiently complementary to nucleotide sequences in either the first genomic strand or the first complementary genomic strand to anneal therewith. Each of the downstream short tail primers has nucleotide sequences which are sufficiently complementary to the nucleotide sequences in the non-complementary primer portion of the downstream long tail primers to anneal therewith but which are not sufficiently complementary to nucleotide sequences in either the first genomic strand or the first complementary genomic strand to anneal therewith, whereby the upstream and downstream short tail primers can be used in the second amplification step selectively to amplify material in duplexes synthesized in the first amplification step and none of the first genomic duplexes.
The inventor, in collaboration with others, has previously shown that PCR amplification of human K-ras gene first exon sequences can be accomplished using an upstream primer (K5') encoding a G.fwdarw.C substitution at the first position of codon 11 (Jiang et al, Oncogene, 4, 923-928 (1989)). The sequence of K5' thus mediates a BstNI restriction enzyme site (CCTGG) overlapping the first two nucleotides of wild-type codon 12. Since this site is absent from mutuant codon 12 fragments, RFLP analysis of the amplified products can be used to detect K-ras oncogenes activated at codon 12. Importantly, a second BstNI site may be strategically incorporated into the downstream primer (K3') as an internal control for enzyme fidelity.
The principle behind the `enriched` amplification procedure of the prior art is described in an article co-authored by the inventor (Kahn et al, (1991). Oncogene, 6, 1079-1083) and shown in the schematic flow diagram of FIG. 1. K-ras first exon sequences are PCR amplified using the upstream primer, K5', and a new downstream primer K3' wt which lacks an internal control BstNI restriction site. The 157 nt long fragment is digested with BstNI, thereby cleaving wild type fragments and rendering them inaccessible for subsequent amplification. The products of the digestion, enriched in full length mutated codon 12 sequences, are then used in a second round of PCR amplification with primers K5' and K3'. These samples are subject to RFLP analysis by digestion with BstNI, followed by polyacrylamide gel electrophoresis of the products.
Referring to FIG. 1, in a first round of amplification (A), primers K5' and K3' wt (wild-type) are utilized for the synthesis of a 157 nt fragment including codon 12 sequences. K5' contains a nucleotide substitution at the first position of codon 11, creating a BstNI restriction site (CCTGG) overlapping the first two nucleotides of wild type codon 12 (hatched box). Digestion of PCR amplified sequences from the first round with BstNI leaves uncleaved products enriched in mutant codon 12 sequences (black box). These uncleaved products are subject to a second round of amplification (B) using primers K5' and K3' (containing a control BstNI site; cross-hatched box). Upon RFLP analysis with BstNI, sequences derived from a mutated codon 12 allele show bands of 143 and 14 nt, while amplified wild type allele remnants are cleaved to generate fragments of 114, 29, and 14 nt.
While the enriched PCR and other techniques discussed above provide approaches for the possible early detection of mutant alleles, there are drawbacks in their use for the identification of a mutant allele in a pre-neoplastic lesion. So, for example, in the enriched PCR technique, although it may be desirable to amplify in a second amplification step only duplexes which were formed in a first amplification step, no procedure has been provided to prevent amplification in a second amplification step of genomic DNA which was present in a test sample originally. Another drawback of the enriched PCR technique and the other prior art techniques discussed above is that they are cumbersome and are not easily adapted for use in diagnostic kits. For example, the previously used method for detection of mutant alleles in the enriched PCR technique involves a gel separation of selectively amplified mutant alleles from others. What has been needed is a more sensitive and less cumbersome method of detection that can be easily converted into a diagnostic kit. What has also been needed is a quantitative procedure to enable quantification of the results of a genetic screening. What has further been needed is a simplification of the three stage procedure of the prior art (involving amplification, digestion, re-amplification and final digestion followed by PAGE analysis).